Molecular ultrasound and MRI of Vascular Development. - Renewal - 1

描述(申请人提供)：基因工程小鼠已被广泛用于血管发育的活体研究，并用于研究中风、癌症和缺血性心脏病等广泛人类疾病潜在的血管变化。因此，迫切需要活体方法来分析小鼠模型发育和疾病过程中三维(3D)血管形态和基因表达模式的动态变化。为了在这一领域取得进展，需要报告鼠进行比传统光学显微镜更具穿透性的血管成像。在之前的资助期间，我们建立了两个新的超声和磁共振成像(MRI)报告系统，这是一种体内方法，可以用于发育中和成年小鼠的高分辨率3D血管成像。具体地说，我们开发了一种用于细胞表面生物素化的“Biotag”转基因小鼠，并利用超声和MRI的亲和型造影剂制备了Tie2-Biotag转基因小鼠，用于血管内皮细胞(VEC)的靶向成像。我们还发现，二价金属转运蛋白DMT1可以作为锰增强磁共振成像(MEMRI)的高效报告基因，MEMRI是一种体内成像方法，可以标记血管内皮细胞和平滑肌细胞(SMC)这两种主要的血管细胞类型。我们现在建议充分利用分子成像技术的这些突破，并建立和验证下一代通用报告小鼠，用于从胚胎到成体阶段的血管形态和基因表达模式的成像。这些报告小鼠将被用来在体内建立野生型(WT)小鼠胚胎和Gli2-/-突变体中发育中的血管系统的分子成像方法，我们发现Gli2-/-突变体在大脑动脉中存在花纹缺陷。我们还将利用成人血管生成的体内模型来测试和验证通用报告小鼠。该项目的具体目标是：1)优化从胚胎到成年阶段血管成像的超声和MRI方案；2)建立一个通用的Biotag报告鼠，用于体内多种VEC基因的多模式表达成像；以及3)建立一个通用的DMT1报告鼠，用于VEC和SMC的体内MEMRI成像。这项研究将产生并验证用于核磁共振和超声波体内血管成像的通用报告小鼠，使从胚胎到成年阶段血管形态和基因表达模式的动态变化能够进行前所未有的研究。

DESCRIPTION (provided by applicant): Genetically-engineered mice have been utilized widely for in vivo studies of vascular development and for studying vascular changes underlying pathogenesis in a wide range of human diseases such as stroke, cancer and ischemic heart disease. As a result, there is a critical need for in vivo methods to analyze dynamic changes in three-dimensional (3D) vascular morphology and gene expression patterns during development and disease in mouse models. To progress in this area, reporter mice are required for vascular imaging with more penetration than conventional optical microscopy. During the previous funding period, we established two novel reporter systems for ultrasound and magnetic resonance imaging (MRI), in vivo methods that can be used for high-resolution, 3D vascular imaging in developing and adult mice. Specifically, we developed a "Biotag" transgene for cell surface biotinylation, and generated Tie2-Biotag transgenic mice for targeted imaging of vascular endothelial cells (VECs) using avidinated contrast agents for both ultrasound and MRI. We also discovered that the Divalent Metal Transporter, DMT1 can be utilized as a highly effective reporter gene for Mn-enhanced MRI (MEMRI), an in vivo imaging method that has the potential for labeling both VECs and smooth muscle cells (SMCs), the two major vascular cell types. We now propose to take full advantage of these breakthroughs in molecular imaging technology, and to generate and validate the next generation universal reporter mice for imaging vascular morphologies and gene expression patterns from embryonic to adult stages. These reporter mice will be used to establish in vivo approaches for molecular imaging of the developing vasculature in wild type (WT) mouse embryos, and in Gli2-/- mutants, which we showed have patterning defects in the cerebral arteries. We will also utilize in vivo models of adult angiogenesis to test and validate the universal reporter mice. The specific aims of the project are: 1) Optimize ultrasound and MRI protocols for vascular imaging from embryonic to adult stages; 2) Establish a universal Biotag reporter mouse for in vivo, multi-modality expression imaging of a variety of VEC genes; and 3) Establish a universal DMT1 reporter mouse for in vivo MEMRI imaging of both VECs and SMCs. This research will generate and validate universal reporter mice for in vivo vascular imaging with MRI and ultrasound, enabling unprecedented studies of dynamic changes in vascular morphologies and gene expression patterns, from embryonic to adult stages.